Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.     

Tissue plasminogen activator is a potent activator of PDGF-CC

Tissue plasminogen activator (tPA) is a serine protease involved in the degradation of blood clots through the activation of plasminogen to plasmin. Here we report on the identification of tPA as a specific protease able to activate platelet-derived growth factor C (PDGF-C). The newly identified PDGF-C is secreted as a latent dimeric factor (PDGF-CC) that upon proteolytic removal of the N-terminal CUB domains becomes a PDGF receptor alpha agonist. The CUB domains in PDGF-CC directly interact with tPA, and fibroblasts from tPA-deficient mice fail to activate latent PDGF-CC. We further demonstrate that growth of primary fibroblasts in culture is dependent on a tPA-mediated cleavage of latent PDGF-CC, generating a growth stimulatory loop. Immunohistochemical analysis showed similar expression patterns of PDGF-C and tPA in developing mouse embryos and in tumors, indicating both autocrine and paracrine modes of activation of PDGF receptor-mediated signaling pathways. The identification of tPA as an activator of PDGF signaling establishes a novel role for the protease in normal and pathological tissue growth and maintenance, distinct from its well-known role in plasminogen activation and fibrinolysis.     

A novel giant gene CSMD3 encoding a protein with CUB and sushi multiple domains: a candidate gene for benign adult familial myoclonic epilepsy on human chromosome 8q23.3-q24.1

We identified a novel giant gene encoding a transmembrane protein with CUB and sushi multiple domains on the human chromosome 8q23.3-q24.1 in which benign adult familial myoclonic epilepsy type 1 (BAFME1/FAME, OMIM:601068) has been mapped. This giant gene consists of 73 exons and spans over 1.2Mb on the genomic DNA region. It showed significant homology to two genes, CSMD1 gene on 8p23 and CSMD2 gene on 1p34, at reduced amino acid sequence level and hence we designated as CSMD3. The CSMD3 gene was expressed mainly in adult and fetal brains. We performed mutation analysis on the CSMD3 gene for seven patients with BAFME1/FAME, but no mutation was found in the coding sequence of the CSMD3 gene. Comparative genomic analysis revealed a conserved family of CSMD genes in the mouse and fugu genomes. Possible functions of the CSMD gene family are discussed.     

Effect of the tyrosine kinase inhibitor lapatinib on CUB-domain containing protein (CDCP1)-mediated breast cancer cell survival and migration

The surface receptor CUB domain-containing protein 1 (CDCP1) is highly expressed in several adenocarcinomas and speculated to participate in anchorage-independent cell survival and cell motility. Tyrosine kinase phosphorylation seems to be crucial for intracellular signaling of CDCP1. Lapatinib, a tyrosine kinase inhibitor (TKI), is approved for treatment of HER-2/neu overexpressing metastatic breast cancer and functions by preventing autophosphorylation following HER-2/neu receptor activation. This study aimed to investigate the effect of CDCP1 expression on anchorage-independent growth and cell motility of breast cancer cells. Moreover, studies were performed to examine if lapatinib provided any beneficial effect on HER-2/neu^(+)/−/CDCP1⁺ breast cancer cell lines. In our studies, we affirmed that CDCP1 prevents cells from undergoing apoptosis when cultured in the absence of cell–substratum anchorage and that migratory and invasive properties of these cells were decreased when CDCP1 was down-regulated. However, only HER-2/neu⁺, but not HER-2/neu^(+)/− cells showed decreased proliferation and invasion and an enhanced level of apoptosis towards loss of anchorage when treated with lapatinib. Therefore, we conclude that CDCP1 might be involved in regulating adhesion and motility of breast cancer cells but that lapatinib has no effect on tyrosine kinases regulating CDCP1. Nonetheless, other TKIs might offer therapeutic approaches for CDCP1-targeted breast cancer therapy and should be studied considering this aspect.     

Identification of the Matriptase Second CUB Domain as the Secondary Site for Interaction with Hepatocyte Growth Factor Activator Inhibitor Type-1

Matriptase is a type II transmembrane serine protease comprising 855 amino acid residues. The extracellular region of matriptase comprises a noncatalytic stem domain (containing two tandem repeats of complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein (CUB) domain) and a catalytic serine protease domain. The stem domain of matriptase contains site(s) for facilitating the interaction of this protease with the endogenous inhibitor, hepatocyte growth factor activator inhibitor type-1 (HAI-1). The present study aimed to identify these site(s). Analyses using a secreted variant of recombinant matriptase comprising the entire extracellular domain (MAT), its truncated variants, and a recombinant HAI-1 variant with an entire extracellular domain (HAI-1–58K) revealed that the second CUB domain (CUB domain II, Cys³⁴⁰–Pro⁴⁵²) likely contains the site(s) of interest. We also found that MAT undergoes cleavage between Lys³⁷⁹ and Val³⁸⁰ within CUB domain II and that the C-terminal residues after Val³⁸⁰ are responsible for facilitating the interaction with HAI-1–58K. A synthetic peptide corresponding to Val³⁸⁰–Asp³⁹⁰ markedly increased the matriptase-inhibiting activity of HAI-1–58K, whereas the peptides corresponding to Val³⁸⁰–Val³⁸⁹ and Phe³⁸²–Asp³⁹⁰ had no effect. HAI-1–58K precipitated with immobilized streptavidin resins to which a synthetic peptide Val³⁸⁰–Pro³⁹² with a biotinylated lysine residue at its C terminus was bound, suggesting direct interaction between CUB domain II and HAI-1. These results led to the identification of the matriptase CUB domain II, which facilitates the primary inhibitory interaction between this protease and HAI-1.     

P2X7 receptor antagonists display agonist-like effects on cell signaling proteins

Background

The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca²⁺]_i. We report that some agents that can block P2X₇R receptors also promote diverse P2X₇R-independent effects on cell signaling.

Methods

We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X₇R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.

Results

With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated ⁴⁵Ca²⁺ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X₇R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.

Conclusions

Several agents used as P2X₇R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.

General significance

Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.     

Plasticity of the TSG-6 HA-binding loop and mobility in the TSG-6-HA complex revealed by NMR and X-ray crystallography

Tumour necrosis factor-stimulated gene-6 (TSG-6) is a glycosaminoglycan-binding protein expressed during inflammatory and inflammation-like processes. Previously NMR structures were calculated for the Link module of TSG-6 (Link_TSG6) in its free state and when bound to an octasaccharide of hyaluronan (HA(8)). Heparin was found to compete for HA binding even though it interacts at a site that is distinct from the HA-binding surface. Here we present crystallography data on the free protein, and (15)N NMR relaxation data for the uncomplexed and HA(8)-bound forms of Link_TSG6. Although the Link module is comparatively rigid overall, the free protein shows a high degree of mobility in the beta4/beta5 loop and at the Cys47-Cys68 disulfide bond, both of which are regions involved in HA binding. When bound to HA(8), this dynamic behaviour is dampened, but not eliminated, suggesting a degree of dynamic matching between the protein and sugar that may decrease the entropic penalty of complex formation. A further highly dynamic residue is Lys54, which is distant from the HA-binding site, but was previously shown to be involved in heparin binding. When HA is bound, Lys54 becomes less mobile, providing evidence for an allosteric effect linking the HA and heparin-binding sites. A mechanism is suggested involving the beta2-strand and alpha2-helix. The crystal structure of free Link_TSG6 contains five molecules in the asymmetric unit that are highly similar to the NMR structure and support the dynamic behaviour seen near the HA-binding site: they show little or no electron density for the beta4/beta5 loop and display multiple conformations for the Cys47-Cys68 disulfide bond. The crystal structures were used in docking calculations with heparin. An extended interface between a Link_TSG6 dimer and heparin 11-mer was identified that is in excellent agreement with previous mutagenesis and calorimetric data, providing the basis for further investigation of this interaction.     

非洲爪蟾孵化酶分子对卵黄膜作用方式的研究

非洲爪蟾的孵化液对卵黄膜和二甲基酷蛋白具有降妥活性。用非洲爪蟾孵化酶的特异性抗ＧＳＴ－ＵＶ．２抗体进行Ｗｅｓｔｅｒｎ杂交的结果表明,孵化液中出现一种分子量为６０ｋＤ的大组分,有时也会出现一种分子量为４０ｋＤ的小组分。     

Constraint on di-nucleotides by codon usage bias in bacterial genomes

It has been reported earlier that the relative di-nucleotide frequency (RDF) in different parts of a genome is similar while the frequency is variable among different genomes. So RDF is termed as genome signature in bacteria. It is not known if the constancy in RDF is governed by genome wide mutational bias or by selection. Here we did comparative analysis of RDF between the inter-genic and the coding sequences in seventeen bacterial genomes, whose gene expression data was available. The constraint on di-nucleotides was found to be higher in the coding sequences than that in the inter-genic regions and the constraint at the 2nd codon position was more than that in the 3rd position within a genome. Further analysis revealed that the constraint on di-nucleotides at the 2nd codon position is greater in the high expression genes (HEG) than that in the whole genomes as well as in the low expression genes (LEG). We analyzed RDF at the 2nd and the 3rd codon positions in simulated coding sequences that were computationally generated by keeping the codon usage bias (CUB) according to genome G+C composition and the sequence of amino acids unaltered. In the simulated coding sequences, the constraint observed was significantly low and no significant difference was observed between the HEG and the LEG in terms of di-nucleotide constraint. This indicated that the greater constraint on di-nucleotides in the HEG was due to the stronger selection on CUB in these genes in comparison to the LEG within a genome. Further, we did comparative analyses of the RDF in the HEG rpoB and rpoC of 199 bacteria, which revealed a common pattern of constraints on di-nucleotides at the 2nd codon position across these bacteria. To validate the role of CUB on di-nucleotide constraint, we analyzed RDF at the 2nd and the 3rd codon positions in simulated rpoB/rpoC sequences. The analysis revealed that selection on CUB is an important attribute for the constraint on di-nucleotides at these positions in bacterial genomes. We believe that this study has come with major findings of the role of CUB on di-nucleotide constraint in bacterial genomes.